A method of measuring the concentration of a fluorescent dye contained in a sample is described in Gary R. Bright, “Multiparameter Imaging of Cellular Function” on pp 204-215 in “Fluorescent and Luminescent Probes for Biological Activity” edited by W. T. Mason, 1993, Academic Press, USA. This method is to extract a wavelength component necessary for excitation of a fluorescent dye, from white light by means of a dichroic filter and to illuminate a sample with the wavelength component to produce fluorescence. This fluorescence is detected through a band-pass filter and with a monochrome (black-and-white) camera, and the intensity thereof is measured. The band-pass filter is used for receiving only the wavelength component corresponding to the fluorescent dye in the sample, with the camera. Since the intensity of the fluorescence emitted from the fluorescent dye in the sample is proportional to a concentration of the fluorescent dye, the intensity of the fluorescence measured in this manner is handled as the concentration of the fluorescent dye.
This method is implemented using a filter set consisting of the dichroic filter and the band-pass filter according to the fluorescent dye. As long as the sample contains only one kind of fluorescent dye, one filter set can be fixedly used. However, if the sample contains two or more kinds of fluorescent dyes, a plurality of filter sets must be used as replaced one from another during measurement of the sample. The replacement of filter set will cause a slight change in an optical system and thus affect the accuracy of the measurement. In addition, the replacement of filter set will produce a difference between measurement times of different fluorescent dyes. This is undesirable for measurement of a living sample.
The above-described method also has a point to be improved in terms of the measurement accuracy. In a case where peak wavelengths of fluorescent dyes in a sample are located close to each other, wings of fluorescence spectra overlap with each other. In this case, it is infeasible to extract only a wavelength component corresponding to a single fluorescent dye from the fluorescence spectra even with the use of the band-pass filter, and a wavelength component of another fluorescent dye shall be mixed in the fluorescence having passed through the filter. This degrades the accuracy of the measurement of the fluorescent dye.